UV mutagenesis of Bacteria

Determination of Optimal UV treatment

This procedure is used to determine optimal treatment which will be used for library generation.

  1. Grow culture overnight as appropriate for strain.
  2. Pellet 1mL of overnight culture at 3k rcf for 5 minutes.
    • see note 1
  3. Aspirate media.
  4. Resuspend in 1mL sterile saline.
  5. Label sterile petri dishes for each of the for each condition to be tested for each strain.
    • see note 2
    • Typical conditions (all in ÁJ/cm2):
      • 0 -- required for determining death rate!
      • 5,000
      • 10,000
      • 15,000
      • 20,000
      • 25,000
      • 30,000
  6. Near the UV crosslinker to minimize contamination concerns, transfer 120Ál of cells to the center of each petri dish. Cells should form a distinct droplet.
  7. Place the dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish.
    • good practice to place the 0 sample in the crosslinker as well.
  8. Transfer 100ÁL from the droplet to a labeled tube some careful pipetting can increase number of cells recovered as cells may settle during experiment. Be careful not to introduce air bubbles.
  9. Plate for ~200 cells assuming 0 death rate.
    • replicate plating or higher number of cells can be used to increase confidence in death rate.
  10. Grow o/n
  11. Calculate death rate based on number of colonies.
    • see note 3
  12. Update following table for future reference.

Table of previously determined optimal (~95-99% death ratios). Please update with additional results.

Strain ID ÁJ/cm2 Strain description Researcher
SKO16 27,500 BWA25113 with plasmid DED -- via SKO
unk 10,000 acenobacter BR

Library Generation

  1. Grow culture overnight as appropriate for strain.
  2. Pellet 1mL of overnight culture at 3k rcf for 5 minutes in each of 4 eppendorf tubes.
  3. Aspirate media.
  4. Resuspend each in 1mL sterile saline.
  5. Label 2 sterile petri dishes with 0 and optimal ÁJ/cm2 treatment for each strain.
  6. Near the UV crosslinker to minimize contamination concerns, transfer a total of at least 11 120Ál droplets of cells distributed around the dish. Typically up to 6 droplets can easily be kept distinct on a single plate.
  7. Place each dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish.
    • good practice to place the 0 sample in the crosslinker as well.
  8. Transfer 100ÁL from each droplet to a labeled tube. Some careful pipetting up and down can lead to recovery of more cells as they may settle during experiment. Take care to not introduce air bubbles.
  9. Pellet cells at 3k rcf for 5 minutes.
  10. Resuspend in appropriate growth media.
  11. Transfer appropriate amount of cells to o/n culture. This can either be a dilution or nearly the entire volume of cells depending on how large of a library you want to start with.
  12. Plate both treated and control for ~200 cells assuming 0 death rate to estimate total number of viable mutants.
    • replicate plating or higher number of cells can be used to increase confidence in death rate.
  13. Grow plate and culture o/n.
  14. Calculate number of mutants based on number of colonies.
    • This is expected to be very similar to previously calculated death ratios x the total number of cells treated.
  15. Freezedowns should be created for o/n culture of viable cells and possibly resuspended treated cells depending on downstream applications.

Notes

  1. Single attempt at using 1/3 of the total concentration of cells did not result in any noticable differences. DED SKO GFP project.
  2. Conceivably 1 petri dish could be used with multiple strains, but risk of contamination obviously higher.
    • This has not been attempted to my knowledge (remove if you attempt with confident results or warning to not attempt in future).
  3. Not suggested to extrapolate kill ratios, best to test additional conditions if 95+% death rate not achieved.

-- Main.DanielDeatherage - 13 Feb 2014

Edit | Attach | Print version | History: r3 < r2 < r1 | Backlinks | Raw View | Raw edit | More topic actions...

 Barrick Lab  >  ProtocolList  >  ProtocolsUVLibrary

Topic revision: r1 - 13 Feb 2014 - 17:37:11 - Main.DanielDeatherage
 
This site is powered by the TWiki collaboration platformCopyright ©2017 Barrick Lab contributing authors. Ideas, requests, problems? Send feedback